The proposed collaborative research on fundamental aspects of sequencing by hybridization (SBH) is intended to lead to a long term productive interaction between the SBH development teams at the Houston Advanced Research Center (HARC) and the Engelhardt Institute of Molecular Biology (EIMB), Russian Academy of Sciences, Moscow. the specific aims of the IGRCP application are: (i) implement a model system for investigating various aspects of SBH, consisting of a DNA chip ("genosensor") targeting a 300-base section of genomic DNA in the human HPRT locus (including exons 7 and 8, the short intron between them, and a small amount of flanking sequence); (ii) prepare a model DNA chip targeted to the 300bp HPRT region, consisting of a nested set of octamers, overlapping by one nucleotide along both strands of the target region, covalently bound to the surface at distinct sites within the chip (thin gel matrix at EIMB and glass surface at HARC); (iii) conduct hybridization experiments using model oligonucleotide "target" strands, followed by second-stage hybridization with appropriate hexamer probes to evaluate the "continuous stacking hybridization" (CSH) strategy that has been proposed by the collaborators at EIMB to solve the problem of sequence repeats in SBH; (iv) perform hybridization with PCR-amplified target-specific genomic DNA, followed by CSH; (v) implement EIMB collaborators' existing software for SBH/CSH in the UNIX/C++ environment; and (vi) perform computer analysis of genosensor-derived hybridization data to acquire an extensive set of data on the efficiency of mismatch discrimination in hybridization, including the influence of nearest neighbors on mismatch stability. The research proposed in this application is a serious attempt to address several of the technical hurdles that must be overcome before SBH technology can be used for total sequence determination. In addition, the model system proposed here would also enable initial feasibility studies of the use of genosensors for polymorphic marker analysis (for genomic mapping and individual identification), for detection of mutations in genes associated with genetic diseases (genetic diagnostics), and for monitoring of mutations arising from chronic, low level exposure of cells to genotoxic agents. Scientific exchange between HARC and EIMB would also facilitate establishment of additional partnerships between genome researchers at EIMB and other research institutions of the former Soviet Union and their U.S. counterparts in academia, industry and government laboratories.